Real-time PCR for mRNA quantitation.

By M. Wong, J. Medrano. BioTechniques, 2005

1,769 citations recorded by the source corpus.

Real-time PCR has become one of the most widely used methods of gene quantitation because it has a large dynamic range, boasts tremendous sensitivity, can be highly sequence-specific, has little to no post-amplification processing, and is amenable to increasing sample throughput. However, optimal benefit from these advantages requires a clear understanding of the many options available for running a real-time PCR experiment. Starting with the theory behind real-time PCR, this review discusses the key components of a real-time PCR experiment, including one-step or two-step PCR, absolute versus relative quantitation, mathematical models available for relative quantitation and amplification efficiency calculations, types of normalization or data correction, and detection chemistries. In addition, the many causes of variation as well as methods to calculate intra- and inter-assay variation are addressed.

Subfields: Molecular Biology

View Paper: https://www.semanticscholar.org/paper/b58ecd8476388c7acdaa7dd654d1aac62f1a5d19

How researchers see this paper

The researchers whose trusted networks rank it highest, strongest advocate first. Every researcher gets a different order.

In Molecular Biology

1. Huajun Wang
2. Athanasia Spandidos
3. C. Prgomet
4. S. Fleige
5. M. W. Pfaffl
6. Xiaowei Wang
7. Brian Seed
8. J. Sehm

Citations in the corpus

• Comparison of relative mRNA quantification models and the impact of RNA integrity in quantitative real-time RT-PCR (2006, background): “With regard to the efficiency of the PCR, those can affect by a number of variables like length of the amplicon, RNA secondary structure and primer quality (Bustin and Nolan 2004; Wong and Medrano 2005 ).”
• Quantitative PCR to estimate copepod feeding (2008, neutral): “standards and quantiWcation is reported as the number of gene copies present (Wong and Medrano 2005).”
• Diagnosing COVID-19: The Disease and Tools for Detection (2020, methodology): “36 This assay format is more sensitive than the one-step assay, but it is more time-consuming and requires optimizing additional parameters.”
• RNA integrity and the effect on the real-time qRT-PCR performance. (2006, background): “Sometimes, even intact RNA does not guarantee good results because RNA sample may contain inhibitors that can reduce reaction efficiency (Bustin and Nolan, 2004a; Wong and Medrano, 2005).”
• Predicted impact of ocean acidification on a marine invertebrate: elevated CO2 alters response to thermal stress in sea urchin larvae (2009, background): “They highlight the importance of looking at multiple environmental factors simultaneously as this approach may reveal previously unsuspected biological impacts of atmospheric changes.”
• Evaluation of DNA microarray results with quantitative gene expression platforms (2006, methodology): “Quantitative, real-time PCR has been developed over the last decade to specifically measure template molecule number”
• PrimerBank: a PCR primer database for quantitative gene expression analysis, 2012 update (2011, background): “Real-time PCR has several major advantages over DNA microarrays, the most important being a large dynamic range, high sensitivity and high specificity (5,6).”
• An overview of calf diarrhea - infectious etiology, diagnosis, and intervention (2014, methodology): “Real-time PCR (as known as quantitative PCR or qPCR) is a method that is capable of not only amplifying the target sequence but also quantifying the amount of target with great sensitivity and high throughput [142].”